Research proposed in the present application represents an extension of studies on the diphtheria toxin repressor (DtxR) that have been performed over the past fourteen years. These studies have culminated in a detailed understanding of the structure function relationships, identification of both the ancillary and primary metal ion binding sites, and a working model of the mechanism by which inactive apo-DtxR transits to its active metal ion bound form. In vitro analysis of the hyperactive mutant DtxR(E175K) has revealed that the apo-repressor is blocked in a "half-activated" state requiring only trace amounts of transition metal ions for full repressor activity. In contrast, in vivo DtxR(E175) maintains a constitutively active phenotype due to the inability to remove sufficient levels of transition metal ions from intact microorganisms while still maintaining viability. We have employed the Positive Selection of DtxR (PSDT) screen to survey random mini-gene libraries for peptides capable of activating the wild type DtxR. Using this screen, we have isolated two classes of Peptide Activators of wild type DtxR, or PADs and demonstrated by cross-linking and other biophysical methods that the PAD's specifically bind and activate members of the DtxR-family of repressors. We have begun to determine the minimal amino acid sequence necessary for PAD induced activation of repressor activity. In addition, we have found that the PADs are bacteriostatic in iron-rich medium and bacteriocidal in iron-depleted medium. Preliminary transcritpome analysis has suggested that PAD activation of target repressors results in the global modulation of metal ion-dependent gene expression and ribosomal gene clusters. Research proposed in this application is based on the hypothesis that PAD induced activation of repressors in the apo-DtxR-superfamily in a low iron in vivo setting results in the loss of metal ion homeostasis and leads to a bacteriocidal response. Accordingly, we propose the peptide activators of the apo-DtxR-family of repressors represent a novel class of antimicrobial agents whose action is based upon the global repression of metal ion sensitive genes in metal ion-deplete environments.